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Biofilms are dense bacterial colonies, derived from microbially derived sessile community, networked within a polysaccharide matrix with a distinct architecture that has the attachment potential to both alive and abiotic surfaces. Pseudomonas aeruginosa is a model biofilm forming microorganism associated with remarkable morbidity and mortality rate due to emergence of antibiotic resistant pathogenic bacteria. Moreover, Pseudomonas aeruginosa originating from a biofilm is more resistant to a wide range of antibiotics than the planktonic bacteria. This research was planned to develop a comparative study of the biofilm production between potential, antimicrobial resistance of Pseudomonas aeruginosa isolated from mature environmental biofilm and clinical strain of the same species that did not derive from biofilm. It was observed that the Pseudomonas aeruginosa from environmental isolates were resistant to 15 prominent antibiotics, while clinical strain was comparatively resistant to only few of them. A confirmatory analysis of biofilm formation and antibiotic resistance pattern of these two groups of organisms was checked by 96-well microtiter plate and the disc diffusion method respectively. Finally, the results portrayed that the environmental strains with high drug resistance, potentially formed a considerable amount of biofilm in the period of a week whereas; clinical stains formed a negligible amount of biofilm within the same time frame.